Mutations in the X-chromosomal AR gene have been identified as the underlying molecular lesion in human androgen insensitivity syndrome (AIS) and understanding of AR structure/function relationships are based upon these naturally occurring mutations. The overall goal of this proposal is to understand how AR structure/function, and the cellular factors that influence AR function, interact to promote AR-mediated action. Recent data regarding phenotypic heterogeneity suggests that AIS comprises a more complex set of regulatory errors influencing AR function. The goals of this research proposal are: 1) To determine whether endogenous cellular factors influence AR transcriptional activity in cultured human genital skin fibroblasts and play a critical role in the determination of sex phenotype; 2) To identify AR gene mutations within exon 1 encoding the N-terminal transcriptional activation domain of AR(or the 5' regulatory region) to determine the functional role of this domain on AR transactivation and AR gene expression, respectively; 3) To determine the nucleotide (s) and their spacing within a novel androgenreceptor-specific, transcriptionally active response element and the amino acids within the DNA-binding domain of the putative coactivators with the human AR steroid-binding and transactivation; and 4) To characterize the interaction of putative coactivators with the human AR steroid-binding domain and to determine if these interactions are disrupted by mutations in AR.